The purpose of this proposal is to provide training that will prepare me for independent biomedical research focused on intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDRs) as well as the roles they play in regulating calcium-triggered synaptic vesicle exocytosis, a fundamental biological process that all neurons in the brain use to communicate. This will be accomplished by investigating the interactions between synaptotagmin-1 (Syt1), a protein that helps confer calcium-sensitivity to exocytosis and contains an IDR, and the SNARE proteins syntaxin-1, SNAP-25, and synaptobrevin, IDPs that assemble into a four-helix bundle that helps promote fusion of vesicle and plasma membranes in order to release vesicle contents into the synaptic cleft. Our understanding of how these proteins cooperate with one another to accomplish this goal has been limited by only studying their atomic structures in the absence of membrane bilayers, the most critical element of their natural cellular environment. This proposal aims to address this limitation by studying the molecular interaction of Syt1 with SNARE proteins while all are anchored to and actively bridging two separate membranes. First, in Aim 1 of this proposal, several nuclear magnetic resonance (NMR) methods will be used to determine the molecular structure of Syt1 by itself anchored to a lipid nanodisc by its transmembrane region in the absence of calcium ion. Importantly, this membrane-anchored Syt1 includes its long 60-amino acid IDR, which has not been stringently studied but may play a role in assisting the Syt1 interaction with SNAREs. This molecular structure will serve as a reference state for the protein at rest. Complementary to Aim 1, Aim 2 will combine NMR methods with cryo-electron microscopy (cryo-EM) to determine the molecular structure of Syt1 bound to the SNARE proteins as they actively bridge two nanodisc membranes, mimicking their arrangement just before a synaptic vesicle fuses with the plasma membrane. This molecular structure will serve as the primed reference state. Finally, the impact of calcium ion on this primed Syt1/trans-SNARE interaction will be determined using NMR and cryo-EM, revealing the molecular changes triggered by calcium that ultimately promote the membrane fusion of exocytosis. These results will significantly enhance our molecular understanding of how neurons release neurotransmitters and will provide an avenue for developing therapeutic strategies of the pre- synapse. Overall, execution of this proposal will equip me with several technical research skills and rigorous quantitative reasoning well suited for continued investigation of IDPs and IDRs and the roles they play in fundamental processes of molecular neuroscience.